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Supplementary tables S1-6. Supplementary table S1 Primers used for RT-qPCR analyses. Supplementary table S2 Clinical variables associated with differences in gene expression among 119 resected liver metastases. Supplementary table S3 613 genes whose expression was significantly upregulated and 481 genes whose expression was significantly (p

Purpose: Chemotherapy treatment of metastatic colon cancer ultimately fails due to development of drug resistance. Identification of chemotherapy-induced changes in tumor biology may provide insight into drug resistance mechanisms.Experimental Design: We studied gene expression differences between groups of liver metastases that were exposed to preoperative chemotherapy or not. Multiple patient-derived colonosphere cultures were used to assess how chemotherapy alters energy metabolism by measuring mitochondrial biomass, oxygen consumption, and lactate production. Genetically manipulated colonosphere-initiated tumors were used to assess how altered energy metabolism affects chemotherapy efficacy.Results: Gene ontology and pathway enrichment analysis revealed significant upregulation of genes involved in oxidative phosphorylation (OXPHOS) and mitochondrial biogenesis in metastases that were exposed to chemotherapy. This suggested chemotherapy induces a shift in tumor metabolism from glycolysis towards OXPHOS. Indeed, chemotreatment of patient-derived colonosphere cultures resulted in an increase of mitochondrial biomass, increased expression of respiratory chain enzymes, and higher rates of oxygen consumption. This was mediated by the histone deacetylase sirtuin-1 (SIRT1) and its substrate, the transcriptional coactivator PGC1α. Knockdown of SIRT1 or PGC1α prevented chemotherapy-induced OXPHOS and significantly sensitized patient-derived colonospheres as well as tumor xenografts to chemotherapy.Conclusions: Chemotherapy of colorectal tumors induces a SIRT1/PGC1α-dependent increase in OXPHOS that promotes tumor survival during treatment. This phenomenon is also observed in chemotherapy-exposed resected liver metastases, strongly suggesting that chemotherapy induces long-lasting changes in tumor metabolism that potentially interfere with drug efficacy. In conclusion, we propose a novel mechanism of chemotherapy resistance that may be clinically relevant and therapeutically exploitable. Clin Cancer Res; 21(12); 2870–9. ©2015 AACR.

Supplementary Figures S1-3. Supplementary figure S1 Cytochrome c oxidase (COX IV) is highly expressed in chemotherapy-treated liver metastases Supplementary figure S2 Expression changes of glycolytic and OXPHOS genes in response to chemotherapy. Supplementary figure S3 Oxygen consumption rates of colonospheres treated with chemotherapy +/- nicotinamide

Objectives To investigate the relevance of lymphangiogenic gene expression in primary and liver metastasis of colorectal cancer (CRC) and identify determinants of lymphatic invasion. Background Lymphatic development promoting vascular endothelial growth factor C (VEGFC) is associated with poor outcome in primary CRC. For colorectal liver metastasis (CRLM), intrahepatic lymph invasion and lymph node metastasis are poor prognostic factors. Exact biological factors promoting lymphatic involvement remain elusive, just as the association with molecular subtypes of CRC. Methods We designed a lymphangiogenic gene set (VEGFC, Nrp-2, PDPN, LYVE-1, MRC1, CCL-21) and applied it to large datasets of CRC. Gene expression of the lymphangiogenic signature was assessed in resected CRLM specimens by Rt-QPCR. In vitro experiments were performed with colon cancer cell line Colo320 (high Nrp-2 expression) and human dermal microvascular lymphatic endothelial cells (LECs). Results Lymphangiogenic gene expression was associated with poor prognosis in both primary and liver metastasis of CRC. CRLM with high expression of consensus molecular subtype-4 identifier genes also exhibited high lymphangiogenic gene expression. Lymph node recurrence following CRLM resection was associated with high expression of VEGFC and Nrp-2. Blocking Nrp-2 significantly reduced invasion of Colo320 cells through an LEC monolayer. Conclusions Lymphangiogenic gene expression is correlated with worse prognosis and consensus molecular subtype-4 in both primary and liver metastatic CRC. VEGFC and Nrp-2 expression may be predictive of lymph node involvement in recurrence after resection of CRLM. Nrp-2, expressed on both tumor and LECs, may have a mechanistic role in lymphatic invasion and is a potential novel target in CRC.

Background and aim Colorectal cancer (CRC) remains one of the leading causes of cancer-related death. Novel therapeutics are urgently needed, especially for tumours with activating mutations in KRAS (∼40%). Here we investigated the role of RAF1 in CRC, as a potential, novel target. Methods Colonosphere cultures were established from human tumour specimens obtained from patients who underwent colon or liver resection for primary or metastatic adenocarcinoma. The role of RAF1 was tested by generating knockdowns (KDs) using three independent shRNA constructs or by using RAF1-kinase inhibitor GW5074. Clone-initiating and tumour-initiating capacities were assessed by single-cell cloning and injecting CRC cells into immune-deficient mice. Expression of tight junction (TJ) proteins, localisation of polarity proteins and activation of MEK-ERK pathway was analysed by western blot, immunohistochemistry and immunofluorescence. Results KD or pharmacological inhibition of RAF1 significantly decreased clone-forming and tumour-forming capacity of all CRC cultures tested, including KRAS -mutants. This was not due to cytotoxicity but, at least in part, to differentiation of tumour cells into goblet-like cells. Inhibition of RAF1-kinase activity restored apicobasal polarity and the formation of TJs in vitro and in vivo , without reducing MEK-ERK phosphorylation. MEK-inhibition failed to restore polarity and TJs. Moreover, RAF1-impaired tumours were characterised by normalised tissue architecture. Conclusions RAF1 plays a critical role in maintaining the transformed phenotype of CRC cells, including those with mutated KRAS . The effects of RAF1 are kinase-dependent, but MEK-independent. Despite the lack of activating mutations in RAF1, its kinase domain is an attractive therapeutic target for CRC.

Background Microvessel density (MVD), as a derived marker for angiogenesis, has been associated with poor outcome in several types of cancer. This study aimed to evaluate the prognostic value of MVD in stage II and III colon cancer and its relation to tumour-stroma-percentage (TSP) and expression of HIF1A and VEGFA. Methods Formalin-fixed paraffin-embedded (FFPE) colon cancer tissues were collected from 53 stage II and 54 (5-fluorouracil-treated) stage III patients. MVD was scored by digital morphometric analysis of CD31-stained whole tumour sections. TSP was scored using haematoxylin-eosin stained slides. Protein expression of HIF1A and VEGFA was determined by immunohistochemical evaluation of tissue microarrays. Results Median MVD was higher in stage III compared to stage II colon cancers (11.1% versus 5.6% CD31-positive tissue area, p

Gene expression-based classification systems have identified an aggressive colon cancer subtype with mesenchymal features, possibly reflecting epithelial-to-mesenchymal transition (EMT) of tumor cells. However, stromal fibroblasts contribute extensively to the mesenchymal phenotype of aggressive colon tumors, challenging the notion of tumor EMT. To separately study the neoplastic and stromal compartments of colon tumors, we have generated a stroma gene filter (SGF). Comparative analysis of stroma(high) and stroma(low) tumors shows that the neoplastic cells in stroma(high) tumors express specific EMT drivers (ZEB2, TWIST1, TWIST2) and that 98% of differentially expressed genes are strongly correlated with them. Analysis of differential gene expression between mesenchymal and epithelial cancer cell lines revealed that hepatocyte nuclear factor 4α (HNF4α), a transcriptional activator of intestinal (epithelial) differentiation, and its target genes are highly expressed in epithelial cancer cell lines. However, mesenchymal-type cancer cell lines expressed only part of the mesenchymal genes expressed by tumor-derived neoplastic cells, suggesting that external cues were lacking. We found that collagen-I dominates the extracellular matrix in aggressive colon cancer. Mimicking the tumor microenvironment by replacing laminin-rich Matrigel with collagen-I was sufficient to induce tumor-specific mesenchymal gene expression, suppression of HNF4α and its target genes, and collective tumor cell invasion of patient-derived colon tumor organoids. The data connect collagen-rich stroma to mesenchymal gene expression in neoplastic cells and to collective tumor cell invasion. Targeting the tumor-collagen interface may therefore be explored as a novel strategy in the treatment of aggressive colon cancer.

The use of bidirectional barbed suture appears to be safe for closing the vaginal cuff in a total laparoscopic hysterectomy and for closing the hysterotomy site during laparoscopic myomectomy., Objective: To evaluate the safety and efficacy of using bidirectional barbed suture in laparoscopic myomectomy (LM) and total laparoscopic hysterectomy (TLH). Methods: This was a case series of clinical outcomes following 172 consecutive LM and TLH cases over a 1-year period conducted at a university teaching hospital. It included 172 women (ages 17 to 81), requiring a myomectomy or hysterectomy for symptomatic uterine fibroids, pelvic pain, or abnormal uterine bleeding; 117 women underwent TLH and 55 women underwent LM. Patients were contacted over the phone 6 months after surgery to inquire about number of days of postoperative vaginal bleeding, visits to the hospital due to bleeding, dyspareunia, and other potential complications. Results: For TLH, the average duration of surgery was 109 minutes, average uterine weight was 256 grams (range, 18 to 1242), and average blood loss was 71mL. In LM, average duration of surgery was 125 minutes, average weight of fibroids was 252g, average number of fibroids removed was 4.0, and average blood loss was 159mL. Seven percent of patients and 8% of their partners had persistent dyspareunia after surgery. There were no conversions to laparotomy. Conclusions: The use of bidirectional barbed suture appears to be safe for closing the vaginal cuff in a TLH and for closing the hysterotomy site during a laparoscopic myomectomy.

Purpose: Chemotherapy treatment of metastatic colon cancer ultimately fails due to development of drug resistance. Identification of chemotherapy-induced changes in tumor biology may provide insight into drug resistance mechanisms. Experimental Design: We studied gene expression differences between groups of liver metastases that were exposed to preoperative chemotherapy or not. Multiple patient-derived colonosphere cultures were used to assess how chemotherapy alters energy metabolism by measuring mitochondrial biomass, oxygen consumption, and lactate production. Genetically manipulated colonosphere-initiated tumors were used to assess how altered energy metabolism affects chemotherapy efficacy. Results: Gene ontology and pathway enrichment analysis revealed significant upregulation of genes involved in oxidative phosphorylation (OXPHOS) and mitochondrial biogenesis in metastases that were exposed to chemotherapy. This suggested chemotherapy induces a shift in tumor metabolism from glycolysis towards OXPHOS. Indeed, chemotreatment of patient-derived colonosphere cultures resulted in an increase of mitochondrial biomass, increased expression of respiratory chain enzymes, and higher rates of oxygen consumption. This was mediated by the histone deacetylase sirtuin-1 (SIRT1) and its substrate, the transcriptional coactivator PGC1α. Knockdown of SIRT1 or PGC1α prevented chemotherapy-induced OXPHOS and significantly sensitized patient-derived colonospheres as well as tumor xenografts to chemotherapy. Conclusions: Chemotherapy of colorectal tumors induces a SIRT1/PGC1α-dependent increase in OXPHOS that promotes tumor survival during treatment. This phenomenon is also observed in chemotherapy-exposed resected liver metastases, strongly suggesting that chemotherapy induces long-lasting changes in tumor metabolism that potentially interfere with drug efficacy. In conclusion, we propose a novel mechanism of chemotherapy resistance that may be clinically relevant and therapeutically exploitable. Clin Cancer Res; 21(12); 2870–9. ©2015 AACR.

Expression of the lymphangiogenic growth factors, in particular VEGF-C, are correlated with disease-free and overall survival in primary colorectal cancer (CRC). Moreover, in patients with metastatic CRC (mCRC) lymphatic invasion and lymph node metastases are correlated with worse outcome, but the biology underlying these correlations remains unknown. To study a potential relationship between tumor biology, lymphatic spread from liver metastases and outcome, we compiled a lymph-endothelium gene set the lymphangiogenesis signature comprised of six genes specifically expressed by lymph endothelial cells, or growth factors stimulating them. In primary colorectal cancer, expression of this signature correlated with increased risk of distant metastasis. When used to cluster a cohort of 119 colorectal liver metastases (CRLM) based on gene expression profiles, the signature identified an aggressive poor prognosis subtype of CRLM with mesenchymal gene expression and a stem-like tumor phenotype. RTqPCR analysis of CRLMs with and without lymph node recurrence during follow up showed that VEGF-C and neuropilin-2 were significantly higher expressed by CRLM with onward spread to the lymph nodes. We conclude that CRLM expressing high levels of lymphangiogenic genes are more likely to develop lymph node metastases during follow up and that this correlates with a general aggressive tumor biology (i.e. the mesemcnymal/stem-like phenotype) and poor survival. Post liver resection adjuvant therapy targeting onward spread to the lymph nodes may be beneficial for patients with mesenchymal-type mCRC. Citation Format: Thomas Vellinga, Dieuwke Marvin, Inne Borel Rinkes, Jeroen Hagendoorn, Onno Kranenburg. Lymphangiogenic gene expression is associated with lymph node recurrence and poor survival after hepatectomy for colorectal liver metastases. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B01.

OBJECTIVE To compare surgical outcomes of laparoscopic myomectomy and robot-assisted laparoscopic myomectomy. METHODS Retrospective cohort study of 115 consecutive laparoscopic myomectomy and 174 consecutive robot-assisted laparoscopic myomectomy performed at Brigham and Women's Hospital over a period of 31 months. Uterine incisions were closed in multiple layers (running barbed suture was used for most cases in the laparoscopic myomectomy group). Surgical outcomes measured included operative time, estimated intraoperative blood loss, length of hospital stay, and perioperative complications. Odds ratios and 95% confidence intervals were calculated from multivariable logistic regression models; adjusted geometric means were estimated from linear regression models on logged outcomes because of skewed distributions. RESULTS Surgical groups were similar in age, body mass index, and leiomyoma characteristics. Robot-assisted laparoscopic myomectomy had significantly longer operative time than laparoscopic myomectomy (adjusted geometric mean of 195.1 compared with 118.3 minutes, P

Study Objective To evaluate and compare recovery times and quality of life (QOL) in patients undergoing a total laparoscopic hysterectomy (TLH) and laparoscopic supracervical hysterectomy (LSH). Design Patients underwent either a TLH or LSH. After surgery, patients maintained a daily log documenting pain, nausea, use of pain medications, and return to daily activities. They also completed a QOL questionnaire (SF-36) before and after surgery. Design Classification Prospective cohort study (Canadian Task Force Classification II-1). Setting University teaching hospital. Patients A total of 122 women undergoing laparoscopic hysterectomy. Measurements and Main Results A total of 122 women underwent TLH (n = 71) or LSH (n = 51) for benign indications from February 2008 to January 2010. There was a significantly higher postoperative improvement of QOL scores in the LSH group in 6 of 10 questionnaire categories and summary scores, including physical functioning (p =.03), role physical (p =.002), and bodily pain (p =.03). There were no significant differences in use of pain medications, level of pain, level of nausea, or return to normal activities. Conclusion LSH appears to provide greater improvement in short-term postoperative QOL compared with TLH. No significant differences were found in postoperative pain or return to daily activities.

To compare perioperative outcomes during laparoscopic myomectomy using a bidirectional barbed suture vs conventional smooth suture.Retrospective analysis of 138 consecutive laparoscopic myomectomies performed by a single surgeon over 3 years (Canadian Task Force classification II-2).Major university teaching hospital.One hundred thirty-eight women with symptomatic uterine myomas.In women undergoing laparoscopic myomectomy from February 2007 through April 2010, conventional smooth sutures were used in 31 patients, and bidirectional barbed suture in 107 patients.The primary indications for laparoscopic myomectomy in either group were pelvic pain or pressure and abnormal uterine bleeding. Use of bidirectional barbed suture was found to significantly shorten the mean (SD) duration of surgery (118 [53] minutes vs 162 [69] minutes; p.05) and reduce the duration of hospital stay (0.58 [0.46] days vs 0.97 [0.45] days; p.05). No significant differences were observed between the 2 groups insofar as incidence of perioperative complications, estimated blood loss, and number or weight of myomas removed during surgery.Use of bidirectional barbed suture seems to facilitate closure of the hysterotomy site in laparoscopic myomectomy.

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We report 2 cases of laparoscopic gynecologic procedures, complicated by small bowel obstruction possibly related to use of a hemostatic agent. The cause was most likely from excess material not incorporated in the hemostatic clot at the site of application. Gentle irrigation and removal of excess material from the site of application is recommended by the manufacturer of FloSeal and may reduce the risk of postoperative adhesion formation and small bowel obstruction.

Despite the rapid advances in laparoscopic surgery in the past 2 decades, the initial entry still accounts for approximately 40% to 50% of laparoscopic complications and should be considered the most dangerous step of a laparoscopic procedure. In this review, the authors share a technique for initial umbilical entry, and provide alternative entry sites in cases where umbilical entry is contraindicated.

Introduction: Three-dimensional cell culture methods are currently not widely used for cancer drug discovery due to the specific imaging demands of the systems. In particular there is a need for well validated robust high throughput methods for image-based assays that can be used for high content screening. Methods: Therefore we have developed an image-based three-dimensional high throughput toxicity assay. We used two platforms, the Thermo Scientific ArrayScan VTi and BD FACSCanto™, to characterize antiproliferative potency, cell death and cell cycle distribution. Cultures of uniform spheroids were treated with Irinotecan either alone or in combination with the ABC transporter inhibitor Valspodar, which blocks irinotecan efflux. All drugs were dispensed using a Tecan HP-D300 digital dispenser. Treatment-induced effects were analyzed after 48, 72 or 96 hours with both platforms using dihydro derivatives of fluorescent live cell dyes such as dihydrocalcein, AM and DNA-binding dyes such as DRAQ5™ and Propidium Iodide. Results: All cell lines tested on the two platforms showed the expected effects of Irinotecan alone or in combination with Valspodar and correlations between the Thermo Scientific ArrayScan VTi and BD FACSCanto™ were high, with Pearson tests varying from 0.76 to 0.95, p < 0.05, depending on the cell line used. All results were reproducible and showed minor variation. Conclusion: The demonstrated approach provides an easy-to-handle, low-cost, accurate and reproducible high content method for measuring drug efficacy in three-dimensional cell cultures. This method can be used for high throughput drug discovery screens and personalized cancer care. Citation Format: Kari Trumpi, David A. Egan, Thomas T. Vellinga, Inne H.M Borel Rinkes, Onno W. Kranenburg. High throughput toxicity assay for three-dimensional cell cultures. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5388. doi:10.1158/1538-7445.AM2014-5388

Background: Altered energy metabolism is one of the hallmarks of cancer. Tumor cells reprogram their energy metabolism to meet the demands of uncontrolled cell division. During tumorigenesis the vast majority of cancer cells become highly glycolytic (Warburg effect) accompanied by a decrease in oxidative metabolism. Chemotherapy is likely to affect the energy metabolism of tumor cells, but how specific drugs affect specific metabolic pathways is only beginning to be addressed. Methods: the effects of cytotoxic agents on energy metabolism were assessed by flow cytometric uptake of Mitotracker®, the ratio of mitochondrial and nuclear DNA on qRT-PCR, western blotting for protein levels of the different complexes of the respiratory chain and oxygen consumption rate by the Seahorse Extracellular Flux Analyzer. Cell death was analyzed by flow cytometric uptake of Propidium Iodide, Nicoletti assay and protein levels of caspases 3 and 8. Results: Gene expression analysis was performed on 119 resected liver metastases of colorectal tumors. Of all clinical variables tested, neoadjuvant chemotherapy was most prominently associated with changes in gene expression. Gene ontology and pathway analysis tools revealed that many of the chemotherapy-associated genes were involved in the regulation of oxidative phosphorylation (OxPhos). To test whether chemotherapy affects OxPhos patient derived colorectal spheroids were treated with the standard cytotoxic agents oxaliplatin and 5-fluorouracil. Chemotherapy strongly increases mitochondrial load, oxygen consumption rate and mitochondrial ATP synthesis. In line with these results chemo-treated tumor cells displayed a higher ratio of mitochondrial-to-nuclear DNA and expression of respiratory complex components was strongly increased following chemotherapy. Chemotherapy strongly induced expression of the histone deacetylase SIRT1, which has been implicated in mitochondrial biogenesis. Inhibition (by either nicotinamide, EX-527, Tenovin-6) or knockdown of SIRT1 prevented the chemotherapy-induced increase in oxidative phosphorylation. Moreover, SIRT1 knockdown greatly reduced tumor cell survival and clonogenic capacity following removal of chemotherapy. Conclusion: Chemotherapy induces an increase in oxidative phosphorylation via SIRT1 and this is required for tumor cell survival following drug removal. Since DNA repair requires ATP, mitochondrial biogenesis may be part of the tumor cell response to DNA-damaging agents. Post-chemotherapy targeting of SIRT1 (or OxPhos enzymes) may be an interesting novel approach to increase chemotherapy efficacy. Citation Format: Thomas T. Vellinga, Vincent C. de Boer, Tijana Borovski, Kari Trumpi, Szabolcs Fatrai, Onno Kranenburg, Inne H.M. Borel Rinkes, Jeroen Hagendoorn. Survival of colorectal cancer cells following chemotherapy relies on a SIRT1-dependent increase in oxidative phosphorylation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3351. doi:10.1158/1538-7445.AM2014-3351

Purpose To determine the surgical and oncologic outcomes after major liver surgery for colorectal liver metastases (CRLM) at a Dutch University Hospital. Patients and Methods Consecutive patients with CRLM who had undergone major liver resection, defined as ≥ 4 liver segments, between January 2000 and December 2015 were identified from a prospectively maintained database. Results Major liver surgery was performed in 117 patients. Of these, 26 patients had undergone formal extended left or right hemihepatectomy. Ninety-day postoperative mortality was 8%. Major postoperative complications occurred in 27% of patients; these adverse events were more common in the extended hemihepatectomy group. Median disease-free survival was 11 months and median overall survival 44 months. Conclusion Major liver surgery, including formal extended hemihepatectomy, is associated with significant operative morbidity and mortality but can confer prolonged overall survival for patients with CRLM.

Highlights • Design of a high throughput toxicity assay for spheroid-type tumor cell cultures. • Cell viability, rather than a proxy thereof, is measured. • Cell viability is measured on a per-cell, rather than per-well, basis. • The method can be implemented on standard automated microscopes. • It provides a low-cost approach for accurate and reproducible toxicity screens., Novel spheroid-type tumor cell cultures directly isolated from patients’ tumors preserve tumor characteristics better than traditionally grown cell lines. However, such cultures are not generally used for high-throughput toxicity drug screens. In addition, the assays that are commonly used to assess drug-induced toxicity in such screens usually measure a proxy for cell viability such as mitochondrial activity or ATP-content per culture well, rather than actual cell death. This generates considerable assay-dependent differences in the measured toxicity values. To address this problem we developed a robust method that documents drug-induced toxicity on a per-cell, rather than on a per-well basis. The method involves automated drug dispensing followed by paired image- and FACS-based analysis of cell death and cell cycle changes. We show that the two methods generate toxicity data in 96-well format which are highly concordant. By contrast, the concordance of these methods with frequently used well-based assays was generally poor. The reported method can be implemented on standard automated microscopes and provides a low-cost approach for accurate and reproducible high-throughput toxicity screens in spheroid type cell cultures. Furthermore, the high versatility of both the imaging and FACS platforms allows straightforward adaptation of the high-throughput experimental setup to include fluorescence-based measurement of additional cell biological parameters.